This article discusses the microsphere-PCR technique, which allows for the amplification of single-stranded DNA using microspheres coated with oligonucleotides. The method enhances specificity and efficiency in DNA amplification processes.
Microsphere-PCR uses a micro-sized sphere coupled to oligonucleotides on their surface, which enables the preferential amplification of single-stranded DNA.
To begin microsphere-PCR, take a reaction mix containing template single-stranded DNA strands, forward primers with an additional nucleotide tag, thermostable DNA polymerase, dNTPs, and microspheres displaying single-stranded oligonucleotide probes.
Place the tube in a thermocycler, and run at the defined conditions.
In the first cycle, at the annealing temperature, the template DNA binds to the probe on the microsphere's surface such that the template DNA functions as the sense strand. During extension, the polymerase extends the DNA, and the duplex remains attached to the microsphere.
In the next cycle, during denaturation, the template DNA separates from the microsphere, leaving the probe strand bound to the surface — serving as a template for a new strand.
During annealing, the tagged primer binds to the antisense strand. Later, the polymerase extends it to synthesize the sense strand with the nucleotide tag.
Repeat the cycle several times to generate multiple copies of sense single-stranded DNA, which go into the solution, while the double-stranded contaminants remain attached.
Transfer the PCR products to a tube containing a loading buffer. Load the product and markers into different wells of the gel, and separate using agarose gel electrophoresis.
The template DNAs form a faint low-molecular-weight band, while an intense high-molecular-weight band confirms single-stranded DNA amplification.
Obtain approximately 25 microspheres through microscopic counting using a light microscope with 40X magnification. Following this, combine all reagents as described in the text protocol. Place the samples into a thermocycler, and start the asymmetric PCR under the appropriate conditions.
The collection of supernatant after a symmetric PCR is one of the most critical steps because it is a major process for generating single-strand DNA with microspheres.
Following amplification, add eight microliters of 6X loading buffer to each sample. Load 15 microliters of each sample into 2% agarose gel. Then, perform electrophoresis at 100 volts for 35 minutes in 1X TAE buffer.
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